Immunol Res (2014) 58:374–377 DOI 10.1007/s12026-014-8511-6
IMMUNOLOGY AT STANFORD UNIVERSITY
Depletion of inflammatory dendritic cells with anti-CD209 conjugated to saporin toxin Michael N. Alonso • Josh G. Gregorio • Matthew G. Davidson Joseph C. Gonzalez • Edgar G. Engleman
Published online: 30 April 2014 Ó Springer Science+Business Media New York 2014
•
Edgar G. Engleman
Abstract Monocytes rapidly infiltrate inflamed tissues and differentiate into CD209? inflammatory dendritic cells (DCs) that promote robust immunity or, if unregulated, inflammatory disease. Previous studies in experimental animal models indicate that inflammatory DC depletion through systemic elimination of their monocyte precursors with clodronate-loaded liposomes ameliorates the development of psoriasis and other diseases. However, translation of systemic monocyte depletion strategies is difficult due to the importance of monocytes during homeostasis and infection clearance. Here, we describe a strategy that avoids the monocyte intermediates to deplete inflammatory DCs through antibody-loaded toxin. Mice with an abundance of inflammatory DCs as a consequence of lipopolysaccharide exposure were treated with antiCD209 antibody conjugated to saporin, a potent ribosome inactivator. The results demonstrate depletion of CD209? DCs. This strategy could prove useful for the targeted reduction of inflammatory DCs in disease. Keywords
Monocytes Inflammatory dendritic cells CD209 Antibody-conjugated toxin Saporin
Introduction Comprising approximately 10 % of circulating leukocytes in humans, monocytes encompass a critical component of both innate and adaptive immunity [1]. During steady state conditions, resident or ‘‘non-inflammatory’’ monocytes replenish tissue macrophages that are essential during wound healing and the clearance of dead or dying cells [2, 3]. During inflammatory responses, monocytes rapidly infiltrate inflamed tissue through CCR2- and CCR5-dependent mechanisms [4, 5]. Upon arrival, monocytes mediate defense against pathogens through the utilization of evolutionarily conserved pattern recognition receptors that recognize and engulf pathogens [6–8]. Inflammatory monocytes also readily Michael N. Alonso and Josh G. Gregorio have contributed equally to this paper. M. N. Alonso J. G. Gregorio M. G. Davidson J. C. Gonzalez E. G. Engleman (&) Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA e-mail: [email protected]
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differentiate into DCs capable of stimulating local immunity or transiting to the draining lymph nodes to stimulate the adaptive immune system [9–12]. Monocytes are essential for the regulation of tissue homeostasis and the control of several pathogens including Listeria monocytogenes, Toxoplasma gondii and Cryptococcus neoformans [6, 13–16]. However, the accumulation of monocyte-derived inflammatory DCs can play an important role in the pathogenesis and persistence of certain inflammatory diseases such as psoriasis and Crohn’s disease [17–21]. As such, broad strategies have been employed to reduce or eliminate monocyte infiltration and subsequent formation of inflammatory DCs. While some of these strategies, such as CCR2 inhibition [22–24] or depletion of phagocytes with clodronate-loaded liposomes [19, 25, 26], have been effective in murine models, they suffer from widespread immune suppression and lack of efficacy in clinical trials [27, 28]. Thus, a new generation of therapeutics is required that more specifically target inflammatory DCs. Recent studies indicate that human and murine inflammatory DCs express CD209 following their differentiation
Stanford Immunology (2014) 58:374–377
from monocytes [11, 20, 21, 29]. As such, we decided to conjugate monoclonal CD209 antibody to the saporin toxin, which is a ribosome inactivating protein that mediates cell death through inhibition of protein synthesis [30]. Saporin is an interesting candidate for targeted cell depletion as it is unable to enter human cells in the absence of a transport protein such as CD209, which mediates phagocytosis upon ligation [31, 32].
Materials and methods Mice C56BL/6 mice were purchased from Jackson Labs. All mice were housed in an American Association for the Accreditation of Laboratory Animal Care-accredited animal facility and maintained in specific pathogen-free conditions. Inflammatory DC formation and toxin administration Six-week-old C56BL/6 mice were injected intravenously with 10 lg of lipopolysaccharide (LPS) (Sigma) to induce inflammatory DC formation and 10 lg of fluorescently conjugated anti-CD209 (eBioscience, Clone 5H10) or isotype control antibody (eBioscience) to label monocytederived inflammatory DCs as described previously [11]. Six hours post injection, mice were injected intravenously with biotinylated anti-CD209 (eBioscience) conjugated to streptavidin–saporin (Advanced Targeting Systems), biotinylated isotype control antibody (eBioscience) conjugated to streptavidin–saporin (Advanced Targeting Systems) or biotinylated anti-CD209 (eBioscience) conjugated to streptavidin–alexa 647 (eBioscience). After 12 h, the inguinal and brachial lymph nodes were extracted and digested for 30 min at 37 °C with 20 U/mL type IV collagenase (Worthington) in RPMI media (Gibco) supplemented with 100 U/mL penicillin, 100 g/mL streptomycin, 2 mM L-glutamine and 10 % fetal calf serum prior to the creation of single-cell suspensions via mechanical dissociation. Flow cytometry Single-cell suspensions were incubated with anti-CD16/32 monoclonal antibody (mAb) (eBioscience) to block Fc receptors prior to staining cells with a panel of mAbs against CD3, CD11b, CD11c, CD19, CD40, DX5, GR1 and MHC II (I-Ab). Cells were washed, labeled with DAPI (Invitrogen) and analyzed on a BD LSR II. FACS plots were generated by FlowJo (Treestar).
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Statistical analysis An unpaired student’s t test (two-tailed) with 95 % confidence interval was utilized to analyze all experimental data. P \ 0.05 was considered significant.
Results Antibody-conjugated toxins deplete inflammatory DCs in vivo To investigate the potential of anti-CD209 antibody conjugated to saporin toxin to deplete inflammatory DCs in vivo, mice were injected intravenously with LPS and fluorescently conjugated anti-CD209 to elicit and label inflammatory DCs, respectively [11, 29]. After 6 h, mice were injected with PBS, biotinylated anti-CD209 conjugated to streptavidin–saporin (CD209-toxin) or biotinylated isotype control antibody conjugated to streptavidin– saporin (iso-toxin). Lymph nodes were processed after 12 h and assessed by flow cytometry. The results indicate that inflammatory DCs, defined as CD209? myeloid DCs (lineage- MHC II? CD11c? CD11b? GR1-), were markedly depleted in a small cohort of mice following administration of CD209-toxin (Fig. 1a). Subsequent experiments in larger cohorts of mice confirmed these results (Fig. 1b). To control for the potential of reduced labeling efficiency of inflammatory DCs in the CD209toxin condition, mice were also injected with biotinylated CD209 conjugated to streptavidin-alexa 647 (CD209Ax647) 6 h after injection of LPS and fluorescently conjugated anti-CD209. The results indicate that the depletion was specific as the frequencies of CD209? DCs were similar between the CD209-Ax647 and iso-toxin conditions (data not shown).
Discussion Monocytes readily infiltrate inflamed tissues and differentiate into inflammatory DCs that are critical for the clearance of infection [9, 10]. However, uncontrolled accumulation of inflammatory DCs can promote and sustain inflammation-associated tissue damage and subsequent depletion of inflammatory DCs through the elimination of monocytes ameliorates disease [19, 25]. In an effort to avoid systemic immunosuppression, we have investigated several methods capable of specifically depleting or inhibiting the formation of inflammatory DCs without eliminating their monocyte precursors. The data presented in this report indicate that saporin-conjugated anti-CD209 antibody efficiently depletes inflammatory DCs in vivo.
123
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Stanford Immunology (2014) 58:374–377
Fig. 1 CD209 conjugated to saporin toxin depletes inflammatory DCs. C57BL/6 mice were given intravenous injections of LPS and antiCD209 to elicit and label inflammatory DCs. After 6 h, PBS, biotinylated anti-CD209 or isotype control antibody conjugated to streptavidin– saporin was administered intravenously. 12 h later, the inguinal and brachial lymph nodes were processed and stained with DC-associated and lineage markers to identify CD209? inflammatory DCs. a Gating strategy utilized to identify lineage- MHC II? CD11c? CD11b? GR1CD209? inflammatory DCs. b Frequency of CD209? inflammatory DCs among myeloid DCs defined as lineage- MHC II? CD11c? CD11b? cells. Data are from three independent experiments and 12 mice (mean and SEM), *P \ 0.05
Conjugation of CD209 to other toxins should also demonstrate significant efficacy at depleting inflammatory DCs in vivo. On a cautionary note, CD209 expression has been reported on splenic pre-plasmacytoid DCs [33]. The effects of antibody-toxin depletion methods on other phagocytes such as monocytes, granulocytes and macrophages warrant further investigations, as these cells may take up antigen– antibody complexes non-specifically. Conflict of interest
The authors declare no conflict of interest.
References 1. Auffray C, Sieweke MH, Geissmann F. Blood monocytes: development, heterogeneity, and relationship with dendritic cells.
123
2.
3. 4.
5.
6.
Annu Rev Immunol. 2009;27:669–92. doi:10.1146/annurev. immunol.021908.132557. Mosser DM, Edwards JP. Exploring the full spectrum of macrophage activation. Nat Rev Immunol. 2008;8(12):958–69. doi:10.1038/nri2448. Gordon S, Taylor PR. Monocyte and macrophage heterogeneity. Nat Rev Immunol. 2005;5(12):953–64. doi:10.1038/nri1733. Weber C, Belge KU, von Hundelshausen P, Draude G, Steppich B, Mack M, et al. Differential chemokine receptor expression and function in human monocyte subpopulations. J Leukoc Biol. 2000;67(5):699–704. Serbina NV, Pamer EG. Monocyte emigration from bone marrow during bacterial infection requires signals mediated by chemokine receptor CCR2. Nat Immunol. 2006;7(3):311–7. doi:10. 1038/ni1309. Serbina NV, Jia T, Hohl TM, Pamer EG. Monocyte-mediated defense against microbial pathogens. Annu Rev Immunol. 2008;26:421–52. doi:10.1146/annurev.immunol.26.021607.0903 26.
Stanford Immunology (2014) 58:374–377 7. Juarez E, Nunez C, Sada E, Ellner JJ, Schwander SK, Torres M. Differential expression of Toll-like receptors on human alveolar macrophages and autologous peripheral monocytes. Respir Res. 2010;11:2. doi:10.1186/1465-9921-11-2. 8. Muzio M, Mantovani A. Toll-like receptors (TLRs) signalling and expression pattern. J Endotoxin Res. 2001;7(4):297–300. 9. Leon B, Lopez-Bravo M, Ardavin C. Monocyte-derived dendritic cells formed at the infection site control the induction of protective T helper 1 responses against Leishmania. Immunity. 2007;26(4):519–31. doi:10.1016/j.immuni.2007.01.017. 10. Wakim LM, Waithman J, van Rooijen N, Heath WR, Carbone FR. Dendritic cell-induced memory T cell activation in nonlymphoid tissues. Science. 2008;319(5860):198–202. doi:10. 1126/science.1151869. 11. Cheong C, Matos I, Choi JH, Dandamudi DB, Shrestha E, Longhi MP, et al. Microbial stimulation fully differentiates monocytes to DC-SIGN/CD209(?) dendritic cells for immune T cell areas. Cell. 2010;143(3):416–29. doi:10.1016/j.cell.2010.09.039. 12. Varol C, Vallon-Eberhard A, Elinav E, Aychek T, Shapira Y, Luche H, et al. Intestinal lamina propria dendritic cell subsets have different origin and functions. Immunity. 2009;31(3):502– 12. doi:10.1016/j.immuni.2009.06.025. 13. Traynor TR, Kuziel WA, Toews GB, Huffnagle GB. CCR2 expression determines T1 versus T2 polarization during pulmonary Cryptococcus neoformans infection. J Immunol. 2000;164(4):2021–7. 14. Jia T, Serbina NV, Brandl K, Zhong MX, Leiner IM, Charo IF, et al. Additive roles for MCP-1 and MCP-3 in CCR2-mediated recruitment of inflammatory monocytes during Listeria monocytogenes infection. J Immunol. 2008;180(10):6846–53. 15. Dunay IR, Fuchs A, Sibley LD. Inflammatory monocytes but not neutrophils are necessary to control infection with Toxoplasma gondii in mice. Infect Immun. 2010;78(4):1564–70. doi:10.1128/ IAI.00472-09. 16. Osterholzer JJ, Curtis JL, Polak T, Ames T, Chen GH, McDonald R, et al. CCR2 mediates conventional dendritic cell recruitment and the formation of bronchovascular mononuclear cell infiltrates in the lungs of mice infected with Cryptococcus neoformans. J Immunol. 2008;181(1):610–20. 17. te Velde AA, van Kooyk Y, Braat H, Hommes DW, Dellemijn TA, Slors JF, et al. Increased expression of DC-SIGN ? IL12 ? IL-18 ? and CD83 ? IL-12-IL-18- dendritic cell populations in the colonic mucosa of patients with Crohn’s disease. Eur J Immunol. 2003;33(1):143–51. doi:10.1002/immu.200390017. 18. Baumgart DC, Metzke D, Schmitz J, Scheffold A, Sturm A, Wiedenmann B, et al. Patients with active inflammatory bowel disease lack immature peripheral blood plasmacytoid and myeloid dendritic cells. Gut. 2005;54(2):228–36. doi:10.1136/gut. 2004.040360. 19. Ward NL, Loyd CM, Wolfram JA, Diaconu D, Michaels CM, McCormick TS. Depletion of antigen-presenting cells by clodronate liposomes reverses the psoriatic skin phenotype in KC-Tie2 mice. Br J Dermatol. 2011;164(4):750–8. doi:10.1111/j.13652133.2010.10129.x. 20. Alonso MN, Wong MT, Zhang AL, Winer D, Suhoski MM, Tolentino LL, et al. T(H)1, T(H)2, and T(H)17 cells instruct monocytes to differentiate into specialized dendritic cell subsets. Blood. 2011;118(12):3311–20. doi:10.1182/blood-2011-03-341065.
377 21. Davidson MG, Alonso MN, Yuan R, Axtell RC, Kenkel JA, Suhoski MM, et al. Th17 cells induce Th1-polarizing monocytederived dendritic cells. J Immunol. 2013;191(3):1175–87. doi:10. 4049/jimmunol.1203201. 22. Rafei M, Berchiche YA, Birman E, Boivin MN, Young YK, Wu JH, et al. An engineered GM-CSF-CCL2 fusokine is a potent inhibitor of CCR2-driven inflammation as demonstrated in a murine model of inflammatory arthritis. J Immunol. 2009; 183(3):1759–66. doi:10.4049/jimmunol.0900523. 23. Gong JH, Ratkay LG, Waterfield JD, Clark-Lewis I. An antagonist of monocyte chemoattractant protein 1 (MCP-1) inhibits arthritis in the MRL-lpr mouse model. J Exp Med. 1997;186(1):131–7. 24. Andres PG, Beck PL, Mizoguchi E, Mizoguchi A, Bhan AK, Dawson T, et al. Mice with a selective deletion of the CC chemokine receptors 5 or 2 are protected from dextran sodium sulfatemediated colitis: lack of CC chemokine receptor 5 expression results in a NK1.1 ? lymphocyte-associated Th2-type immune response in the intestine. J Immunol. 2000;164(12):6303–12. 25. Oelzner P, Kunze A, Henzgen S, Thoss K, Hein G, Stein G, et al. High-dose clodronate therapy prevents joint destruction in chronic antigen-induced arthritis of the rat but inhibits bone formation at the axial skeleton. Inflamm Res. 2000;49(8):424–33. 26. Tacke F, Ginhoux F, Jakubzick C, van Rooijen N, Merad M, Randolph GJ. Immature monocytes acquire antigens from other cells in the bone marrow and present them to T cells after maturing in the periphery. J Exp Med. 2006;203(3):583–97. doi:10.1084/jem.20052119. 27. Haringman JJ, Gerlag DM, Smeets TJ, Baeten D, van den Bosch F, Bresnihan B, et al. A randomized controlled trial with an antiCCL2 (anti-monocyte chemotactic protein 1) monoclonal antibody in patients with rheumatoid arthritis. Arthritis Rheum. 2006;54(8):2387–92. doi:10.1002/art.21975. 28. Vergunst CE, Gerlag DM, Lopatinskaya L, Klareskog L, Smith MD, van den Bosch F, et al. Modulation of CCR2 in rheumatoid arthritis: a double-blind, randomized, placebo-controlled clinical trial. Arthritis Rheum. 2008;58(7):1931–9. doi:10.1002/art.23591. 29. Davidson MG, Alonso MN, Kenkel JA, Suhoski MM, Gonzalez JC, Yuan R, et al. In vivo T cell activation induces the formation of CD209(?) PDL-2(?) dendritic cells. PLoS ONE. 2013; 8(10):e76258. doi:10.1371/journal.pone.0076258. 30. Weng A, Bachran C, Fuchs H, Krause E, Stephanowitz H, Melzig MF. Enhancement of saporin cytotoxicity by Gypsophila saponins–more than stimulation of endocytosis. Chem Biol Interact. 2009;181(3):424–9. doi:10.1016/j.cbi.2009.07.007. 31. Bachran C, Sutherland M, Heisler I, Hebestreit P, Melzig MF, Fuchs H. The saponin-mediated enhanced uptake of targeted saporin-based drugs is strongly dependent on the saponin structure. Exp Biol Med. 2006;231(4):412–20. 32. Zhang P, Schwartz O, Pantelic M, Li G, Knazze Q, Nobile C, et al. DC-SIGN (CD209) recognition of Neisseria gonorrhoeae is circumvented by lipooligosaccharide variation. J Leukoc Biol. 2006;79(4):731–8. doi:10.1189/jlb.0405184. 33. O’Keeffe M, Hochrein H, Vremec D, Caminschi I, Miller JL, Anders EM, et al. Mouse plasmacytoid cells: long-lived cells, heterogeneous in surface phenotype and function, that differentiate into CD8(?) dendritic cells only after microbial stimulus. J Exp Med. 2002;196(10):1307–19.
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IMMUNOLOGY AT STANFORD UNIVERSITY
Depletion of inflammatory dendritic cells with anti-CD209 conjugated to saporin toxin Michael N. Alonso • Josh G. Gregorio • Matthew G. Davidson Joseph C. Gonzalez • Edgar G. Engleman
Published online: 30 April 2014 Ó Springer Science+Business Media New York 2014
•
Edgar G. Engleman
Abstract Monocytes rapidly infiltrate inflamed tissues and differentiate into CD209? inflammatory dendritic cells (DCs) that promote robust immunity or, if unregulated, inflammatory disease. Previous studies in experimental animal models indicate that inflammatory DC depletion through systemic elimination of their monocyte precursors with clodronate-loaded liposomes ameliorates the development of psoriasis and other diseases. However, translation of systemic monocyte depletion strategies is difficult due to the importance of monocytes during homeostasis and infection clearance. Here, we describe a strategy that avoids the monocyte intermediates to deplete inflammatory DCs through antibody-loaded toxin. Mice with an abundance of inflammatory DCs as a consequence of lipopolysaccharide exposure were treated with antiCD209 antibody conjugated to saporin, a potent ribosome inactivator. The results demonstrate depletion of CD209? DCs. This strategy could prove useful for the targeted reduction of inflammatory DCs in disease. Keywords
Monocytes Inflammatory dendritic cells CD209 Antibody-conjugated toxin Saporin
Introduction Comprising approximately 10 % of circulating leukocytes in humans, monocytes encompass a critical component of both innate and adaptive immunity [1]. During steady state conditions, resident or ‘‘non-inflammatory’’ monocytes replenish tissue macrophages that are essential during wound healing and the clearance of dead or dying cells [2, 3]. During inflammatory responses, monocytes rapidly infiltrate inflamed tissue through CCR2- and CCR5-dependent mechanisms [4, 5]. Upon arrival, monocytes mediate defense against pathogens through the utilization of evolutionarily conserved pattern recognition receptors that recognize and engulf pathogens [6–8]. Inflammatory monocytes also readily Michael N. Alonso and Josh G. Gregorio have contributed equally to this paper. M. N. Alonso J. G. Gregorio M. G. Davidson J. C. Gonzalez E. G. Engleman (&) Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA e-mail: [email protected]
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differentiate into DCs capable of stimulating local immunity or transiting to the draining lymph nodes to stimulate the adaptive immune system [9–12]. Monocytes are essential for the regulation of tissue homeostasis and the control of several pathogens including Listeria monocytogenes, Toxoplasma gondii and Cryptococcus neoformans [6, 13–16]. However, the accumulation of monocyte-derived inflammatory DCs can play an important role in the pathogenesis and persistence of certain inflammatory diseases such as psoriasis and Crohn’s disease [17–21]. As such, broad strategies have been employed to reduce or eliminate monocyte infiltration and subsequent formation of inflammatory DCs. While some of these strategies, such as CCR2 inhibition [22–24] or depletion of phagocytes with clodronate-loaded liposomes [19, 25, 26], have been effective in murine models, they suffer from widespread immune suppression and lack of efficacy in clinical trials [27, 28]. Thus, a new generation of therapeutics is required that more specifically target inflammatory DCs. Recent studies indicate that human and murine inflammatory DCs express CD209 following their differentiation
Stanford Immunology (2014) 58:374–377
from monocytes [11, 20, 21, 29]. As such, we decided to conjugate monoclonal CD209 antibody to the saporin toxin, which is a ribosome inactivating protein that mediates cell death through inhibition of protein synthesis [30]. Saporin is an interesting candidate for targeted cell depletion as it is unable to enter human cells in the absence of a transport protein such as CD209, which mediates phagocytosis upon ligation [31, 32].
Materials and methods Mice C56BL/6 mice were purchased from Jackson Labs. All mice were housed in an American Association for the Accreditation of Laboratory Animal Care-accredited animal facility and maintained in specific pathogen-free conditions. Inflammatory DC formation and toxin administration Six-week-old C56BL/6 mice were injected intravenously with 10 lg of lipopolysaccharide (LPS) (Sigma) to induce inflammatory DC formation and 10 lg of fluorescently conjugated anti-CD209 (eBioscience, Clone 5H10) or isotype control antibody (eBioscience) to label monocytederived inflammatory DCs as described previously [11]. Six hours post injection, mice were injected intravenously with biotinylated anti-CD209 (eBioscience) conjugated to streptavidin–saporin (Advanced Targeting Systems), biotinylated isotype control antibody (eBioscience) conjugated to streptavidin–saporin (Advanced Targeting Systems) or biotinylated anti-CD209 (eBioscience) conjugated to streptavidin–alexa 647 (eBioscience). After 12 h, the inguinal and brachial lymph nodes were extracted and digested for 30 min at 37 °C with 20 U/mL type IV collagenase (Worthington) in RPMI media (Gibco) supplemented with 100 U/mL penicillin, 100 g/mL streptomycin, 2 mM L-glutamine and 10 % fetal calf serum prior to the creation of single-cell suspensions via mechanical dissociation. Flow cytometry Single-cell suspensions were incubated with anti-CD16/32 monoclonal antibody (mAb) (eBioscience) to block Fc receptors prior to staining cells with a panel of mAbs against CD3, CD11b, CD11c, CD19, CD40, DX5, GR1 and MHC II (I-Ab). Cells were washed, labeled with DAPI (Invitrogen) and analyzed on a BD LSR II. FACS plots were generated by FlowJo (Treestar).
375
Statistical analysis An unpaired student’s t test (two-tailed) with 95 % confidence interval was utilized to analyze all experimental data. P \ 0.05 was considered significant.
Results Antibody-conjugated toxins deplete inflammatory DCs in vivo To investigate the potential of anti-CD209 antibody conjugated to saporin toxin to deplete inflammatory DCs in vivo, mice were injected intravenously with LPS and fluorescently conjugated anti-CD209 to elicit and label inflammatory DCs, respectively [11, 29]. After 6 h, mice were injected with PBS, biotinylated anti-CD209 conjugated to streptavidin–saporin (CD209-toxin) or biotinylated isotype control antibody conjugated to streptavidin– saporin (iso-toxin). Lymph nodes were processed after 12 h and assessed by flow cytometry. The results indicate that inflammatory DCs, defined as CD209? myeloid DCs (lineage- MHC II? CD11c? CD11b? GR1-), were markedly depleted in a small cohort of mice following administration of CD209-toxin (Fig. 1a). Subsequent experiments in larger cohorts of mice confirmed these results (Fig. 1b). To control for the potential of reduced labeling efficiency of inflammatory DCs in the CD209toxin condition, mice were also injected with biotinylated CD209 conjugated to streptavidin-alexa 647 (CD209Ax647) 6 h after injection of LPS and fluorescently conjugated anti-CD209. The results indicate that the depletion was specific as the frequencies of CD209? DCs were similar between the CD209-Ax647 and iso-toxin conditions (data not shown).
Discussion Monocytes readily infiltrate inflamed tissues and differentiate into inflammatory DCs that are critical for the clearance of infection [9, 10]. However, uncontrolled accumulation of inflammatory DCs can promote and sustain inflammation-associated tissue damage and subsequent depletion of inflammatory DCs through the elimination of monocytes ameliorates disease [19, 25]. In an effort to avoid systemic immunosuppression, we have investigated several methods capable of specifically depleting or inhibiting the formation of inflammatory DCs without eliminating their monocyte precursors. The data presented in this report indicate that saporin-conjugated anti-CD209 antibody efficiently depletes inflammatory DCs in vivo.
123
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Stanford Immunology (2014) 58:374–377
Fig. 1 CD209 conjugated to saporin toxin depletes inflammatory DCs. C57BL/6 mice were given intravenous injections of LPS and antiCD209 to elicit and label inflammatory DCs. After 6 h, PBS, biotinylated anti-CD209 or isotype control antibody conjugated to streptavidin– saporin was administered intravenously. 12 h later, the inguinal and brachial lymph nodes were processed and stained with DC-associated and lineage markers to identify CD209? inflammatory DCs. a Gating strategy utilized to identify lineage- MHC II? CD11c? CD11b? GR1CD209? inflammatory DCs. b Frequency of CD209? inflammatory DCs among myeloid DCs defined as lineage- MHC II? CD11c? CD11b? cells. Data are from three independent experiments and 12 mice (mean and SEM), *P \ 0.05
Conjugation of CD209 to other toxins should also demonstrate significant efficacy at depleting inflammatory DCs in vivo. On a cautionary note, CD209 expression has been reported on splenic pre-plasmacytoid DCs [33]. The effects of antibody-toxin depletion methods on other phagocytes such as monocytes, granulocytes and macrophages warrant further investigations, as these cells may take up antigen– antibody complexes non-specifically. Conflict of interest
The authors declare no conflict of interest.
References 1. Auffray C, Sieweke MH, Geissmann F. Blood monocytes: development, heterogeneity, and relationship with dendritic cells.
123
2.
3. 4.
5.
6.
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